Natural killer T (NKT) cells, co-expressing a T cell receptor and NK cell markers, are essential for several aspects of immunity, such as immunomodulation and immunopotentiation, in various immune diseases including autoimmune diseases, infectious diseases, cancer, etc. NKT cells exist at high levels in the thymus, the liver, and the bone marrow, but at low levels in the spleen, lymph nodes and blood.
Unlike conventional T cells that recognize small peptide antigens presented by major histocompatibility complex MHC class 1 or MHC class 2. NKT cells recognize glycolipid antigens presented by CD1d, a MHC class 1-like molecule. A major subset of NKT cells, called type 1 NKT cells or invariant natural T (iNKT) cells, express an invariant natural T cell receptor (TCR) composed of Vα14-Jα18 chains in mice (Vα24-Jα18 in humans). Upon TCR stimulation with a ligand, such as α-galactosylceramide (α-GC), iNKT cells rapidly produce a wide range of cytokines including IL-4, IFN-γ, L-12, and GM-CSF. This rapid and potent response to a ligand enables iNKT cells to enhance or regulate the activity of various immune cells in innate and acquired immunity. Found in diverse diseases and promoting tumor rejection or regulating autoimmune disorders, these immunomodulatory roles of iNKT cells are studied for use in immunotherapy treatments for cancer and autoimmune diseases.
However, iNKT cells tend to greatly decrease in responsiveness following repeated stimulation after a first stimulation with their ligands via the T cell receptor. For instance, iNKT cells that have been stimulated in vivo with a-GC have reduced proliferation and cytokine production upon secondary stimulation with the same ligand. This iNKT cell anergy is a major obstacle in immunotherapeutic trials targeting iNKT cells.
Conventional T cells are known to become anergic when they receive a TCR signal with insufficient co-stimulatory signals. Co-stimulatory molecules such as CD28, CD40L and ICOS are known to be involved in the development and activation of iNKT cells. Recently, it has been reported that 4-1BB contributes to promote the activation of iNKT cells as a co-stimulatory molecule and can affect iNKT cell-mediated allergic lung inflammation (Kim, D. H., W. S. Chang, Y. S. Lee, K. A. Lee, Y. K. Kim, B. S. Kwon, and C. Y. Kang. 2008. 4-1BB engagement co-stimulates NKT cell activation and exacerbates NKT cell ligand-induced airway hyperresponsiveness and inflammation. J Immunol 180:2062-2068.). On the other hand, it has recently been suggested that coinhibitory molecules, such as PD-1. B7H3, and B7H4, may actively anergize or tolerize T cells by delivering inhibitory signals into TCR-stimulated T cells. In a lymphocytic choriomeningitis virus (LCMV) infected model, CD8 T cells are tolerized by LCMV epitope-presenting dendritic cells. However, the blockade of the PD-1 signal can reverse the anergic phenotype of CD8 T cells. It has also been reported that the inhibition of PD-1/PD-L1 restores the function of exhausted CD8 T cells in a chronic infection model.
Programmed death-1 (PD-1) is a 55 KDa type 1 transmembrane protein of the immunoglobulin superfamily, and is known as a co-inhibitory molecule on T cells. That is, PD-1 is a member of the co-inhibitory molecules of the CD28 family (e.g., CD28, CTLA4, ICOS and BTLA) expressed on activated B cells, T cells and bone marrow cells. Two ligands for PD-1, PD-L1 and PD-L2, have been identified thus far. The interaction of PD-1 with the PD ligands can transduce inhibitory or co-stimulatory signals into the T cells. In conventional T cells, PD-1 is not expressed on naive T cells, but is inducibly expressed after T cell activation. As for PD-L1, it is expressed to some degree on naive T cells and its level is increased on activated T cells. PD-L1 is found at high levels in various human cancers and interacts with PD-1 to transduce inhibitory or co-stimulatory signals from entering into the T cells. For example, the interaction between PD-1 and PD-L1 induces a decrease in the level of tumor invasive lymphocytes and in T cell receptor-mediated proliferation and causes the immune evasion of tumor cells. In PD-1-deficient animals, PD-1 develops various autoimmune phenotypes, such as autoimmune cardiac infarction and lupus-like syndromes with arthritis and nephritis, and plays an important role in the development of autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes and rheumatic arthritis. Aged PD-1-deficient mice develop autoimmune diseases, indicating that PD-1 plays a critical role in the regulation of autoimmunity and immune tolerance. In particular, PD-1 signals are essential for inducing T cell exhaustion during chronic infection.
With the ability thereof to stimulate the T cell receptor to rapidly produce various cytokines of iNKT cells, the NKT cell ligand α-GC has conventionally been used as an anticancer agent. However, since repeated stimulation of iNKT cells with α-GC induces anergy leading to a great decrease in responsiveness, it cannot achieve effective anticancer effects. Therefore, there is a pressing need for an anticancer agent that can restore the responsiveness of iNKT cells even in the state of anergy caused by stimulation with iNKT cell ligands.
Leading to the present invention, intensive and thorough research into an anticancer agent taking advantage of the responsiveness of iNKT cells, conducted by the present inventors, resulted in the finding that PD-1 expressed on iNKT cells is upregulated after stimulation and that blocking of the PD-1/PD-L1 signaling pathway by an anti-PD-1 or anti-PD-L1 antibody allows iNKT cells under an iNKT cell ligand-induced anergy condition to recover their responsiveness, such as the production of cytokines. Also, the anti-PD1 or anti-PD-L1 antibody was found to induce potent anti-tumor activity of iNKT cells as demonstrated by a significant reduction in the number of metastatic nodules in B16F10 melanoma metastasis models in vivo.